Method of recovering and refining cardiolipin



Dec. 21, 1948.

'M.'. PANGBQRN METHODl OF RECOVERING AND REFINING CARDIOLIPIN Filed March 30, 1946 3 Sheets-Sheet 1 JUL.

ATTORNEY Dec. 21, 1948.

M. c. PANGBORN METHOD 0F. RECOVERING AND REFINING CARDIOLIPIN Filed March 50, 1946 ing-2 EMM E16.l l 'l 3 Sheets-Sheet 2 4 E THER PPI (d JELTUE 1 00N VERT 70 Nw .mr /N Erf/ER INVENTOR Marg fmggfrn/ ATTORNEY Dec. 21, 1948. M. CQPANGBORN 2,456,836

METHOD 0F RECOVERING AND REFINING GARDIOLIPIN Filed March so, 1946 s sheets-sheet s (BY Pflss STEPS) 1 (0A/:EN wirf 7j0 18 sMf/a Wzl/ME Vary ,Pm/@gigant EXW/m1 am v ATTORNEY any of the prior teachings.

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Patented Dec. 21, 1948 METHQDOF RECOVERING AND BEFINING io'ARDioi-iirm .Mary C. PangbornfAlbany, N. Y., assigner to the People, of Athe S tateoff-,New York Application March 30, 1946, Serial No. 658,567

2 Claims.

The present invention relates to a method of recovering and refining -cardiolipin, a phospholipid found incertain-animal tissue, especially beef hearts. Some time ago,-it lwas discovered that certain alcoholic extracts of'beef hearts had qualities comparableto that of antigens in that they could be used in i certain serological tests for syphilis. For some time it was not known what material or materials in orA characterictics of the beef heart extract was effective in this respect. Further work, however, was done by the present applicant, which has `shown that a previously unknown phospholipid, Awhich has been isolated from beef heart and purified, iseffective in this use in conjunction with purified lecithin and cholesterol.

This new phospholipid has been given the name cardiolipin Several articles have been published by the present applicant, describing vthe use and certainprior methodsl ofpurifying this inaterial, these articles being published in the following publications: 137A J. Biol. Chem. 545 (1941); 48 Proc. Soc. Exp. Biol. & `Med.V 484v (1941) A14:3 J. Biol. Chem. 24T (1942); 153 'J.\'Biol.' Chem. 34:3 (1944); and 157 J. Biol.- Chem. 691 (-194.5). All of these articles, however, whiley disclosing certain data as to the usefulness of this material Aand some few of the steps of the, present method for the purification thereof, did not kdisclose the present improved process or method of purifying this material, which is set forth hereinafter and which results in the production of a highly .purified forni of cardiolipin, with a yield substantially greater for a given amount of original material used than A primary object of the present invention, therefore, is to provide such a process of recovering and refining cardiolipin as will result in a highly puriiied material `of maximum utility in makingthe tests aforesaid and wherein the lyield is a vmaximum that can be o-btained by any presently known process and with a minimum -of difliculty'in thepractice of the process itself.

Basically the processis onefin--which beef heart muscle is extracted with a suitable solvent and the cardiolipin contained in the extractvmaterial is separated .by Arepeated 1 precipitation with inorganic salts and purified by the use of lorganic solvents.

Cardiol-ipin is known-to occur in other animal tissues than-beef hearts such, for example, as liver. Assuch, the present methodv is notv limited in its application to Ibeefy hearts-but maybe applied to anyy animal tissue containing sufcient amounts of cardiolipin to war-rant theexpenseand 1x1-some in- 2 stances, certain phases of the process may-require some variation in treating other materials .than beef hearts, although the .present process is completely/,operative on such other types of raw material with .the reservation that the efficiency thereof may be `somewhat less using suchother raw or startingmaterials than using beef hearts.

To assist the reader in following the process, there is submitted herewith in the accompanying drawings a now sheet showing the sequenceof steps inthe process and showing a by-pass portion for recovering a relatively small portion of cardiolipin, which would otherwise be lost and which may be recovered and returned to the processby using a special procedure eective on this portion.

In the drawings:

Figs. land 2 combined, when placed together with Fig. 2 below and in juxtaposition toFig. l, illustrate a complete flow sheet of a preferred process in accordance with the present invention; and

vFig. 3 is a similar View of a flowsheetof a by-pass-portion of the process.

`According to my present invention, fresh-beef hearts are mechanically freed'from fat and corinectivetissue as far as isfeasible, then minced or comminuted to a desired extent. This comn minuted tissue is then extracted, preferably at least twice, with a water-miscible organic solvent for fat in which cardiolipin is insoluble, for example; acetone, the acetone being usedin the proportion oi about 1.2 m1. of such solvent per gram of mincedtissue. The solvent and any material dissolved therein `are then removed as by iiltration. This is step I of the accompanying flow sheet.

.The tissue may then be substantially dried and pulverized as step 2. However, if thetissue was finely comminutedin the wet state, as by van emcient homogenizer, it is not necessary to pulvern iZe and the procedure is thereby simplified. Drying to remove acetone is then optional; an alternative procedure is to wash the. tissue on the filter with a small amount of alcohol (ethyl or methyl).

The dried pulverized tissue is then extracted, preferably at least twice, with a lower aliphatic alcohol, preferably methyl alcohol (or ethyl alcohol), preferably in the proportion of about 2 liters of solvent to 300 grams of substantially dried tissue or 2 liters per kilogram of original wet weight. In the preferred process according to the invention, as illustrated by step 3, cach such extraction is carried out at about room teinperature for a period of about 5 to 7 days.

I also contemplate that dried beef heart powder, such as is now available on the market for the preparation of crude antigens, might be substituted for fresh beef hearts, although probably with a lower efliciency. In this extraction it is preferred to use methyl alcohol as it has -been found to be a more efficient solvent for cardiolipin than ethyl alcohol.

Following the extraction with alcohol, the alcohol-insoluble material is discarded, as cardiolipin in the form in which it occurs in the animal tissue, which is probably as the sodium or potassium salt, is reasonably soluble in the lower aliphatic alcohols.

The next step in the process, numbered 4 on the accompanying drawings, is the precipitation of an alkaline earth salt of cardiolipin, preferably by adding a concentrated aqueous solution of an alkaline earth halide such as barium chloride. This treatment is continued until no further precipitate forms. The precipitate is collected, preferably by centrifugation, after the mixture has been allowed to stand for about 24 hours at a temperature of about 36.C. The solid material resulting from this separation and which includes the relatively impure barium salt of cardiolipin is then carried to the next step, numbered 5 on the drawings.

According to some processes for purifying `cardiolipin, this precipitation may be done by cadmium chloride; but in such event the following steps must be varied somewhat as cadmium chloride also precipitates lecithin, which is also present in the original material. Calcium chloride may, however, be used as an alternative which is the preferred to the barium chloride, material for use in this process.

Following this step of the process, the supernatant liquid is separated from the precipitate and may be treated for the recovery of the leci;

thin, which may be selectively precipitated useful adjunct thereto, as the lecithin so prepared metal salt of cardiolipin, such as those of sodium or potassium. I have found that this change from the insoluble alkaline earth salt to the soluble alkali metal salt may be eiected in the presence of a small amount of methyl or ethyl alcohol by adding to the ether solution of the barium salt a 5% aqueous solution of an alkali metal salt such as sodium sulphate. Subsequent alcohols are miscible with either the water phase or the ether phase of the system present.

In particularly carrying out step 5 of the process, the precipitate from the previous step 4 salt into an. alcohol-soluble salt, which is preferably an alkaliV which is wet with methyl alcohol, is treated with about four times its Volume of ether and about 50 ml. of 5% aqueous solution of NazSOi for each ml. ether. The insoluble material, consisting primarily of barium sulphate together with some organic impurities, may then be removed by decantation or preferably by centrifugation. This insoluble material and the aqueous layer are separated and discarded. The ethereal solution, containing the sodium salt of cardiolipin, may then be dried on anhydrous sodium sulphate.

The next step oi the process, numbered 6 on the accompanying drawings, is to concentrate the dried ethereal solution substantially to dryness to eliminate the ether therefrom. Due to the fact that the sodium-salt of cardiolipin is sensitive to deterioration by contact with the oxygen of air and possibly also by light when not in an alcohol solution, the nal stage of this evaporation is done in vacuo in the presence of an inert gas, such as carbon dioxide, and under non-oxidizing conditions, carbon dioxide being used to displace air during all vacuum distillations in the process throughout, so as to protect the material against damage by oxidation. i The next step, numbered I on the accompanying now sheet, may be considered optional, but is usually desirable at thispoint in the process. The object is to remove any remaining water and fats,

step 6 b-y the use of a solvent in which the sodium salt of cardiolipin is insoluble. Such a solvent is, for example, acetone. Particularly this step is done by extracting with several portions of the solvent, preferably warmed to not more than about 50 C. and discarding the solvent and extract.

The next step of the process, numbered 8, is to dissolve the resulting substantially solid material in ether and then convert it to an alcohol solution by adding about 10 volumes (in respect to the ether C. P. absolute methyl alcohol. Any methyl alcohol-insoluble material is discarded at this point, after it has been thoroughly washed with chloride and the mixture u 3-6 C. overnight. This results in two fractions: (a) the supernatant liquid, and (b) a precipitate which I choose to designate as fraction Me-2, the treatment of which will be hereinafter considered as a by-pass portion of the process. Both these fractions contain cardiolipin, but the supernatant liquid contains much more proportionately than The supernatant liquid is then separated from the precipitate in any suitable manner, preferably by simple decantation.V

As step 0 of the process, the cardiolipin in this liquid is precipitated therefrom by adding an of preferably about a 20 0 solution of barium chloride, this solution being added until no further precipitate forms. The mixtureis then chilled in ice, with frequent shakingto aid flocculation.

refrigerated at about This; alkaline earth metal saltofcardiolipin, such as the barium precipitate,y is then collected `by centrifugation, Washed Withmethyl alcohol and preferably also With acetone.

As shown in step l l, this precipitateY is dissolved in ether, C. P. anhydrous, and reprecipitated from the ethereal solution byadding at least an equal volume of acetone. Acetone used in this and subsequentsteps should be of C. P. grade. During this reprecipitation process substances other than cardiolipin are removed in the etheracetone supernatant liquid. It is this step of the process to which the barium salt of cardiolipinresulting from the by-pass series of steps for the purication of the Nie-2 fraction is returned as hereinafter set forth.

The barium salt of cardiolipin forms gelatinous solutions in anhydrous ether, With a characteristic appearance which is helpful in judging the success cf the purication. This reprecipitation by dissolving in ether and reprecipitating. by adding acetone may be repeated the desired number of times until the supernatant liquid is nearly colorless. As a nal step, the reprecipitation may be effected by dissolving in Wet ether or ether to which a little Water is added. In Wet ether clear solution rather than a gel is obtained. The final precipitate obtained from this solution, by adding about two volumes of acetone, is the nearli7 pure barium salt of cardiolipin.

The combined barium salts of cardiolipin prepared as above set forth are then converted to soluble alkali metal salts by dissolving the Ba salt in Wet ether containing about 10% by volume of a lower alpihatic alcohol, such as ethyl or methyl, and the solution shaken vigorously for about five minutes with about one-third its volume of a one-half saturated aqueous solution of sodium chloride cr some other alkali metal soluble salt, preferably a halide. This effects the conversion of the barium salt of cardiolipin to the corresponding sodium or alkali metal salt thereof, as indicated on the accompanying flow sheet `at step l2. The aqueous layer is then drawn off and the. process repeated at least twice again with fresh portions of the aqueous solution of sodium chloride, each time alcohol being added in the proportion of about ten per cent of the ethereal solution present. I have found that the reaction does not go Ato substantial completion unless alcohol is present. The resulting sodium salt of cardiolipin in the ethereal solution may then be treated to remove most of the added alcohol by Washing .it With further portions of one-half saturated aqueous solution of sodium chloride and then drying it on anhydrous sodium sulphate. The ethereal solution is sodium sulphate concentrated by distillation and then poured into about ten volumes of absolute ethyl alcohol. This alcoholic solution may then be concentrated in vacuo to remove ether and clarified by centrifugation, solids being discarded. There results a solution in alcohol of the sodium salt of cardiolipin. It has been found that the solubility of this sodium salt which is now approximately pure, is about 10 Ing/ml. The volume of the solvent used should be so chosen in View of this solubility that all the cardiolipin present will be in solution. This conversion to an alcohol solution of the sodium salt is shown in the accompanying ow sheet as step i3.

Final purification of the cardiolipin may be effected as shown in steps lil' to lli, by precipitatingv the cadmium salt of cardiolipin, for example by adding an aqueousfsolution of CdClz until no then filtered from the further precipitate forms. Itis advantageous to, use a concentrated solution of CdCl'z', for example, 50% by Weight,sinceonly a .small volume of such a solution-is required andthe final mixturesls therefore not. substantiallyl dilutedlwithwater. rThe mixture is then refrigerated to Afacilitate iloccula'tion` and leave a` clear supernatant. The precipitate so formed may'becollected by centriiugationand Washed once with,y absolute ethyl alcohol. This is shown as step lll.

This alcohol-Wet precipitate is thenvmixed with an equal volume of ether which amountsy to .washing the Cd salt with a 1:1 mixture of alcohol and ether.- An excess of ether is to be avoidedfas. it

ill tend to dissolve vthe Cd salt, Which'will then be lost in the supernatant; The. mixture is cen-4 trifuged, the supernatant discarded, and to the precipitate is added sufficient C. P. anhydrous etherto form ya fairly stiff, substantially trans-v parent gel, to which is then gradually added about an equal volume of acetone, This effects the reprecipitaton of the cadmium salt of cardiolipin and. may be repeated one or more times, this being step l5! This purified cadmiumsalt should be substantially clear and colorless in solution in afsmall volume of Wet ether and be finally reprecipitated therefrom by the use of aboutZ to 3 volumesl of acetone.

The final step, numbered it, is-the conversion of the vcadmiumsalt-to the sodium salt which is effected substantially as described for steps l2 and i3 hereinabove, in which thek barium salt is converted to the sodium salt. There results a sodiuml salt of cardiolipin dissolved in absolute ethyl alcohol, which is the final product. This product may then be used in a manner forming nopart of the present invention in preparing antigens; The concentration vof cardiolipin in this solution may be quite accurately determined byperforming a microgravimetric analysis for phosphorus, the sodium salt of cardiolipin con` taining about 4.18 per centvphosphorus, so that the concentration may then be calculated, using this'flgure. v

A satisfactory preparation oi the sodiumsalt of cardiolipin should have the followingproperties:

Solubility-readily soluble in petroleumether, ether or chloroform; moderately soluble'in absolute ethyl alcohol; emulsiable in water. Concentrated solutions shouldappear substantially colorless. The absolute ethanol stock solution is usuallyfcloudy when iirst-prepared, but on standing at 3-6 to settle out to leave a clear'stable solutionwhich may be decanted.`

Analysis; Iodinelnumberz or higher. Nitrogen:` not greater than 0.05 per-cent, and. probably zero in the absolutely pure product.

Returning now to the by-pass procedure,` which is used for recovering relatively'small amounts of cardiolipin found in the Me-Z fractionfrom step El, as shown on the drawings, and particularly referring to Fig. 3, this precipitate resulting from this step andv Which contains thev relatively impure sodium salt of cardiolipinis rst redissolved in methyl alcohol andthe solution acidiiied with an aqueous solution -of a mineral acid such as hydrochloric acid, this step *beingv designated l'l.

The resulting material is then concentrated toA small volume in vacuo to remove most ofthe'l vmethyl alcohol and to leave-an aqueous suspen` C. the trace of insoluble mattertendssion of the desired material plus some impuriconverting the precipitate thus formed to an ties, this being step i8 on the drawing. alcohol-soluble sodium salt of cardiolipin in the The remaining material is then extracted with presence of alcohol by dissolving it in ether and petroleum ether and the aqueous solution readding an aqueous solution of sodium sulphate,

maining after this extraction discarded. The the resultant barium petroleum ether solution is then concentrated sulphate and the aqueous liquid, concentrating substantially to dryness to remove water and eX- the liquid ether phase substantially to dryness cess acid as hydrochloric acid, the cardiolipin in an atmosphere of carbon dioxide, removing at this time being n the folnl of the free acid, remaining fats and water by extracting the rethiS Step beine Shown at i9 in the drawing. The lo sulting material with acetone in which the s'oresidue is preferably freed from Water by adding dium salt of cardiolipin is substantially insoluble a small -portion of absolute alcohol and again and discarding the acetone extract, dissolving evaporating Substanlally to dryness in Vamothe acetone-insoluble residue in ether and con- The residue from this concentration Step 1S verting it to a solution in methyl alcohol by addthen extracted Wltll acetone u1 Which the aoid 15 ing a predetermined. volume of methyl alcohol form of cardiolipin is soluble, any insoluble resiand removing the ether b Vacuum die -Hefe due being discarded toeliminate further iInDuradding a concentrated aqieous solutioiiiJ1 of lgs-i illes, 13h15 bemg step 20. In this step, the acetone dium chloride and maintaining the resultant ma- 1S used preferably 1n '0110 13101301 tion oi about 10 terials at a temperature above but approaching ml. per gram of the solid material to be extracted s thereby. The mixture is warmed to about 50 C. 0 the freezing point for and vigorous shaking used. The dissolved material is then chilled in ice to eliminate substances insoluble in acetone at the lower temperature. The extraction may be repeated with several fresh portions of acetone, so as to recover a maximum yield of the cardiolipin.

The acetone solution is then concentrated nearly to dryness so as to remove excess acetone sairlgdrlnlgu sgnerteds "o ltone in which the barium salt of cardiolipin is diluted with 95% alcohol which is preferably 11150111101?, Separating and discarding the liquid ethyl alcohol but may also be methyl alcohol, from this precipitate, reprecipitating the barium this being step 2l shown in the drawing. sa1t-of ardlohpm a plurality f times by dis' The mm1 Step 22 of this by pass procedure is` l solving. in ether and precipitating it therefrom the preparation of the alcohol-insoluble barium by adomig an exooss of acetone: ooiiVei'i/iiig this salt of cardiolipin, which is effected by neutralizrepiieoipli'ai'ed barium Soit of odidioiipil into a ing the alcohol solution with an aqueous solution Sodium Soii' thereof by dissolving the barium Salt of barium hydroxide. in this connection, other in einen adding ethyl a100h01 and an aqueous alkaline earth metal hydroxides may be a1terna 40 solution of sodium chlori-de and substantially rea predetermined time to precipitate a portion thereof, separating this precipitated portion from the remaining liquid, precipitating from the remaining liquid an alcoholand water-insoluble barium salt of cardiotively employed to Obtain the Corresponding ab. moving the ether by distillation and preparing an kaiineeartn sans of cardiolipin. The precipitate alcohol SOiuuOn 0f the S0d1um Salt 0f cardiolipin, thus formed is separated from the supernatant precipitating the cadmium salt of cardiolipin liquid and added to the corresponding material from this a1001i01 Solution by adding an aqueous entering step il as aforesaid. Since acid car- Solution of oadnfliuIn Chloride llilelelo and `Soloadioiipinis unstable, the entire bypsss procedure, rating the resultant precipitate of the alcoholfrom the vaddition of mineral acid through and insoluble cadmium salt of cardiolipin from the including neutralization with Ba(OH)2, must be liquid, diSSolVing this precipitate in ether and completed within a single day. reprecipitatine it therefrom by adding a sub- Whiie there is not speeifreany disclosed herein stantial amount of acetone in which the cadmium any provision for solvent recovery, it is e0ntem salt of cardiolipin is insoluble and separating and plated that suitable provisions to this end might diSoaIding one liquid, and dissolving the resultant well be made in any commercial operations on Purified Cadmium Sali'I in einer and Converting a substantial scale and based upon solely economic il? to 17110 form 0f an a1o01101-So1ub1e Sodium Salt factors 55 of cardiolipin in the presence of ethyl alcohol by While there is described herein a preferred adding such alcohol and an aqueous solution of process in accordance with the present invention, Sodium Chloride, and preparing an alcohol soluand certain variants of diierent steps thereof tion of the sodium salt of cardiolipin as 'the final are suggested or specically taught, I do not wish Dioduoito be limited except by the scope of the ap- 2. The lorooeSS of recovering and rening pended claims, which are to be construed validly cardiolipin Wilioll comprises the Steps 0f removas broadly as the state of the prior art permits. ing fat and water from comminuted fresh beef What is claimed is: hearts by treating such material with acetone in 1. The process of recovering and rening proportion oi about 1-2 mi poi' gram oi Soiid cardiolipin, which comprises the steps of rematerial, removing the acetone by ltration and moving fat and water from `comminuted beef disoai'diiig i111@ soiuiiioil drying the remaining hearts containing cardiolipin by extracting such i S01i'd material and eXtIaCting it With 95% methyl material with acetone and discarding the exa1o01101 in the proportion of about 2 liters Per 300 tracted material, extracting cardiolipin from the grams diied tissu-o and discarding the S01id Tesiremaining solid material with methyl alcohol due, precipitating cardiolipin from the solution and discarding the solid residue, precipitating by adding a 20% aqueousbarum Chloride soluthe cardiolipin from the alcohol extract solution tion thereto and I'efrigeratng for about 24 hours as an insoluble salt by treating such solution with al? about 3 to 6 C., Separating and discarding `an aqueous solution of barium chloride and the adnliXed liquid, Converting l'le preoloitate separating and discarding the admixed liquid, k thu-s formed to a sodium salt of cardiolipin by dissolving it in ether and shaking with about half the volume of a 5% aqueous scduim sulphate solution in the presence of methyl alcohol, separating and discarding the solid material and the aqueous layer from the ether solution of the sodium salt of cardiolipin, drying the ether solution with anhydrous sodium sulphate, concentrating the ether Solution substantially to dryness under non-oxidizing conditions and in the presence of CO2, extracting the dried material with acetone and discarding the solution to remove remaining fats and Water, dissolving this residue in ether and converting it to a solution in methyl alcohol by adding about 10 volumes of the latter and thereafter distilling in vacuo to remove the ether, adding about 2% by volume of a saturated aqueous solution of sodium -chloride and refrigerating at about 3-6 C. for about 12 to 24 hours to give a liquid fraction and a solid fraction, both containing cardiolipin, separating these fractions and treating the liquid fraction by precipitating the barium salt of cardiolipin therefrom by adding thereto a 20% aqueous solution of barium chloride, separating the precipitate thus formed from and discarding the liquid and then dissolving the precipitate in anyhydrous ether, reprecipitating the barium salt of cardiolipin therefrom by adding at least an equal volume of acetone, repeatedly reprecipitating the barium salt of cardiolipin by dissolving it in ether and reprecipitating it therefrom with acetone until the supernatant liquid is nearly colorless, dissolving the barium salt of cardiolipin in Wet ether containing about 10% by volume ethyl alcohol and adding thereto about one-third the total, volume of a half saturated aqueous solution of sodium chloride to convert the barium salt of cardiolipin into the corresponding sodium salt thereof, drying the ether solution on sodium sulphate, removing the ether by distillation, adding ethyl alcohol to dissolve the cardiolipin salt and precipitating the cadmium salt of cardiolipin from this alcohol solution by adding thereto a concentrated aqueous solution of cadmium i, said by redissolving this solid fraction in 10 chloride, separating the liquid from this precipitate, dissolving this precipitate in ether and reprecipitating therefrom by adding an equal volume of acetone and repeating this procedure a second time, dissolving this precipitate in wet ether and converting it to the sodium salt of cardiolipin in the presence of ethyl alcohol by adding such alcohol and an aqueous solution of sodium chloride and preparing an alcohol solution of the sodium salt of cardiolipin as the iinal product; and recovering cardiolipin from the solid fraction aforesaid obtained by adding 2% by volume of NaCl to the methyl alcohol solution and combining it thereafter in the process aforemethyl alcohol and acidifying it with about 10% of its volume of dilute hydrochloric acid, concentrating the resultant material, including the acid form of cardiolipin in vacuo to remove most of the methyl alcohol, extracting the remaining material with petroleum ether and discarding the aqueous solution, concentrating the petroleum ether solution substantially to dryness in vacuo to remove water and hydrochloric acid, dissolving the acid form of cardiolipin present in acetone and discarding the acetone-insoluble residue, concentrating this acetone solution substantially to dryness in vacuo and dissolving the cardiolipin therefrom with 95% ethyl alcohol, and precipitating the barium salt of cardiolipin therefrom by neutralizing with an aqueous solution of barium hydroxide, and adding this precipitated barium salt to the barium salt aforen said so as to save this portion of the cardiolipin to the process.

MARY C. PANGBORN.

REFERENCES CITED The following references are of record in the nie of this patent: 

